A major component of barrier function in stratified squamous epithelia is the cornified cell envelope (CE). This is a multi-component 10 nm thick layer of highly insoluble protein deposited on the inner surface of the plasma membrane of the cells during terminal differentiation. In the case of the epidermis, a 5 nm thick layer of ceramide lipids (lipid envelope) is attached to the exterior surface. The insolubility of the protein envelope is due in large part to the cross-linking of several structural by transglutaminases. Studies on the biology and assembly of the protein and lipid components are a major effort of this laboratory. Specifically, we are studying: (i) the cross-linking of proteins in CEs isolated from a variety of sources to explore which proteins are cross-linked together through which glutamines and lysines, and to provide information on structure and function; (ii) several key structural proteins such as loricrin, the small proline rich protein (SPR) families, involucrin, envoplakin and periplakin; (iii) the ceramide lipids which become covalently attached to the CE; (iv) the earliest stages of CE assembly produced in cultured keratinocytes; and (v) an attempt to recreate a CE-like structure using an in vitro synthetic lipid vesicle (svi) model system. During FY04, several projects addressing CE structure and assembly were completed (see below). Further research in this area will be continued by Dr L Marekov in association with the LSBR, NIAMS. (1) Co-assembly of Envoplakin and Periplakin into Oligomers and Ca++ dependent Vesicle Binding: Plakin family members envoplakin and periplakin are part of the cornified cell envelope in terminally differentiating stratified squamous epithelia. In FY04, we completed an investigation of the properties and interactionsof purified recombinant human envoplakin and periplakin. We found that envoplakin was insoluble at physiological conditions in vitro, and co-assembly with periplakin was required for its solubility. Envoplakin and periplakin formed soluble complexes with equimolar stoichiometry. Chemical cross-linking revealed that the major soluble form of all periplakin constructs and of envoplakin/periplakin rod domains was a dimer, while co-assembly of the full-length proteins resulted in formation of higher order oligomers. Electron microscopy of rotary-shadowed periplakin demonstrated thin flexible molecules with an average contour length of 88 nm for the rod-plus-tail fragment, and immunolabeling EM confirmed the molecule as a parallel, in-register, dimer. Both periplakin and envoplakin/periplakin oligomers were able to bind synthetic lipid vesicles whose composition mimicked the cytoplasmic side of the plasma membrane of eukaryotic cells. This binding was dependent on anionic phospholipids and Ca++. These findings raise the possibility that envoplakin and periplakin bind to the plasma membrane upon elevation of intracellular [Ca++] in differentiating keratinocytes, where they serve as a scaffold for cornified cell envelope assembly. (2) Effects of knocking-out trichohyalin. This project was initiated in LSB two years ago. During FY04, we designed and constructed the knock-out vector and contarcted to have the ES cells screened and injected into mice. The chimera mice were born and transferred to our collaborator, Dr. Allen Li at Oregon Health & Science University for further characterization. A total of 7 homogyzous mice were generated. Their first coat of hair showed a wavy phenotype initially which fits our prior hypothesis that trichohyalin is needed to strengthen hair shafts. These hairs, however, developed normally after two weeks. Over a longer timescale, the KO mice started to develop hair loss at about 6 months. Also their hair shaft appeared less strong as compared to wildtype shaft. More detailed biochemistry and EM analyses will be conducted to study the effects of trichohyalin loss in hair and other tissues such as skin and tongue. 3)Cellular interactions of periplakin have been characterized by immunofluorescent analysis in cultured human keratinocytes and by in vitro experiments. Its N-terminal domain turned out to be responsible for binding to filamentous actin, while C-terminal domain showed selective binding to keratins 8 and 14, components of intermediate filaments network in simple and stratified epithelia correspondingly. Elongated shape of periplakin molecule revealed by electron microscopy should allow it to efficiently interconnect actin microfilament and the intermediate filament networks, playing role in cell network integration. 4) Role of epiplakin as a cytolinker. Epiplakin is a member of the plakin family with multiple copies of the plakin repeat domain (PRD). We studied the subcellular distribution and interactions of human epiplakin by immunostaining, overlay assays, and RNAi knockdown. Epiplakin decorated the keratin IF network and partially that of vimentin. In the binding assays, the repeat unit (PRD plus linker) showed strong binding and preferentially associated with assembled IF over keratin monomers. Epiplakin knock-down revealed disruption of IF networks in simple epithelial but not in epidermal cells. In rescue experiments, the repeat unit was necessary to prevent the collapse of IF networks in transient knock-down; however, it could only partially restore the keratin but not the vimentin IF network in stably knocked-down HeLa cells. We infer that epiplakin is a versatile cytolinker with functions involved in maintaining the integrity of IF networks in simple epithelial cells. Furthermore, we observed an increase of epiplakin expression in keratinocytes after the calcium switch suggesting the involvement of epiplakin in the process of keratinocyte differentiation. (5) Experiments done in collaboration with an investigator at Jefferson Medical College, showed that caspase 6, which is activated during apoptosis, specifically cleaves periplakin close to C-terminus, effectively separating its intermediate filaments binding part from domain which is responsible for actin binding. This project was completed in FY04.